Death from cancer, especially melanoma, is most often related to metastasis. As the quantitative detection of rare cancer cells in the blood circulation appears to be an early marker of metastatic development, cancer recurrence, and therapeutic efficacy, and as assays for detecting metastatic cells are currently only in vitro techniques that are inadequate to these tasks, our proposal has as its ultimate goal the development of photoacoustic (PA) flow cytometry (FC) in vivo, a new method for ultrasensitive real-time, label-free noninvasive quantitative detection of circulating melanoma cells in blood flow. We will pursue this goal through accomplishment of the following Specific Aims: Aim 1. Develop an advanced PA flow cytometer (PAFC) and verify its parameters in vitro. A new, advanced PA flow cytometer will be developed that uses a near- infrared (IR) laser with a high pulse repetition rate. Its main characteristics, especially its sensitivity threshold, will be evaluated in vitro with the use of static and flowing melanoma cells. Aim 2. Estimate the capability of PAFC to detect single melanoma cells in real time in vivo in an animal model. The absorption contrast of melanoma cells in relation to blood and skin tissues will be determined by PA spectroscopy in vivo in a mouse model at different laser wavelengths. The capability of PAFC for real-time, label-free detection of melanoma cells will be verified by the injection of cells into the tail veins of mice. Aim 3. Ascertain the capability of PAFC to monitor circulating metastatic melanoma cells at different stages of tumor development in an animal model. Metastatic tumor cells will be detected in real time with the PA technique in blood circulation in an animal model at different stages of tumor development, with available conventional assays serving as independent controls. Aim 4. Translate PAFC to human application for quantitative monitoring of CTCs at different stages of melanoma development. The capability of a painless, non-invasive, label-free, fiber- based PAFC for quantitative detection of circulating melanoma cells in vivo in humans will be assessed in four stages: (1) ex vivo PA study of blood samples from healthy donors spiked with melanoma human cell lines; (2) ex vivo PA study of blood samples from melanoma patients verified with conventional assays; (3) in vivo study of healthy individuals as a control group with different skin pigmentation; and 4) melanoma patients at different stages of disease. In the course of this study, we will obtain statistically significant data that will demonstrate this innovative technique's unprecedented capability for quantitatively monitoring circulating melanoma cells in vivo without the need for labeling. The benefits to the public health of achieving this goal extend to routinely monitoring circulating tumor cells as early marker for the micrometastasis development and cancer recurrence in vivo in melanoma patients, as well as to evaluating the efficacy of therapy.